PROJECT SUMMARY/ABSTRACT Interneurons are a diverse class of inhibitory neurons that play a particularly important role in the stability of neural circuits. Defects in interneuron development are associated with epilepsy, autism, bipolar disorder, schizophrenia and other complex neuropsychiatric diseases that have a strong genetic and developmental component. Therefore, elucidating the molecular mechanisms regulating interneuron development is crucial for understanding how the brain operates in both health and disease. Embryonic brain structures in the ventral telencephalon known as the medial, caudal, and lateral ganglionic eminences (MGE, CGE, and LGE, respectively) give rise to interneurons, which migrate to numerous structures throughout the brain including the cortex, hippocampus, amygdala, striatum, and olfactory bulb. While studies that have focused on the genetic specification of ganglionic eminence (GE) progenitors have identified a number of genes critical for interneuron development, currently our understanding of how these populations are specified is far from complete. Even the question of whether distinct or shared progenitor types produce these different subclasses of interneurons remains unanswered. Within each GE there may be genetically distinct but spatially intermixed progenitors with restricted fate potential dedicated to producing particular interneuron types. At the other extreme, a single progenitor type may give rise to multiple interneuron types in a stochastically or temporally controlled manner. Therefore, this proposal is focused on determining what genetic programs regulate interneuron subtype specification and diversity: within the proliferative zones of the ganglionic eminences (addressed in Aim 1) and as interneurons migrate throughout the brain during development (addressed in Aim 2). To accomplish these aims, a transformative high-throughput single-cell RNA sequencing (scRNA-seq) method called Drop-seq will be combined with genetic fate mapping techniques to elucidate the developmental trajectories of gene expression of interneurons from early mitotic progenitors to mature cell types. A preliminary Drop-seq analysis was performed, and the single-cell transcriptomes of more than 10,000 progenitor cells from the MGE, CGE and LGE were sequenced. Heterogeneity across thousands of individual GE progenitors was observed, including differentially expressed transcription factors between the MGE and CGE. As this research continues, additional subpopulation structures across progenitor cells will likely be revealed. Ultimately, this information will be used to construct a cladistic tree of interneuron differentiation, which can be used to infer critical stages and influences on interneuron development. Doing so will play an important role in understanding and ultimately treating the numerous neuropsychiatric diseases associated with interneuron dysfunction.